GENERATION OF INDUCED PLURIPOTENT STEM (iPS) CELLS

ABSTRACT

The present invention relates to a method of generating an induced pluripotent stem (iPS) cell comprising the step of introducing into a target cell one or two coding sequences each giving rise upon transcription to a factor that contributes to the reprogramming of said target cell into an induced pluripotent stem cell and selected from Oct3/4 or a factor belonging to the Myc, Klf and Sox families of factors, wherein the target cell endogenously expresses at least the factors that are not encoded by the coding sequences to be introduced and selected from Oct3/4 or factors belonging to the Myc, Klf and Sox families of factors, and wherein the cell resulting from the introduction of the one or two coding sequences expresses the combination of factor Oct3/4 and at least one factor of each family of factors selected from the group of Myc, Klf and Sox. Furthermore, the present invention relates to an induced pluripotent stem cell generated by the method of the invention and a method of identifying a compound that contributes to the reprogramming of a target cell into an induced pluripotent stem cell. Also, a method of generating a transgenic non-human animal and a composition comprising an iPS cell generated by the method of the present invention for gene therapy, regenerative medicine, cell therapy or drug screening are envisaged.

The present invention relates to a method of generating an inducedpluripotent stem (iPS) cell comprising the step of introducing into atarget cell one or two coding sequences each giving rise upontranscription to a factor that contributes to the reprogramming of saidtarget cell into an induced pluripotent stem cell and selected fromOct3/4 or a factor belonging to the Myc, Klf and Sox families offactors, wherein the target cell endogenously expresses at least thefactors that are not encoded by the coding sequences to be introducedand selected from Oct3/4 or factors belonging to the Myc, Klf and Soxfamilies of factors, and wherein the cell resulting from theintroduction of the one or two coding sequences expresses thecombination of factor Oct3/4 and at least one factor of each family offactors selected from the group of Myc, Klf and Sox. Furthermore, thepresent invention relates to an induced pluripotent stem cell generatedby the method of the invention and a method of identifying a compoundthat contributes to the reprogramming of a target cell into an inducedpluripotent stem cell. Also, a method of generating a transgenicnon-human animal and a composition comprising an iPS cell generated bythe method of the present invention for gene therapy, regenerativemedicine, cell therapy or drug screening are envisaged.

Several documents are cited throughout the text of this specification.The disclosure content of the documents cited herein (includingmanufacturer's specifications, instructions, etc.) is herewithincorporated by reference.

Pluripotent stem cells like embryonic stem (ES) cells are hallmarked bytheir ability to self-renew and differentiate into a wide variety ofcell types. ES cells can be differentiated in vitro into specializedcell lineages of all three embryonic germ layers—ectodermal, mesodermaland endodermal—in the presence of physical inducing and biologicalinducing factors. So far, many promising studies have shown thetherapeutic potential of differentiated derivatives of ESCs inameliorating a range of disease in animal models. As a result,pluripotent stem cells have enormous potential for use in tissueengineering and transplantation therapy. If these cells can be inducedto differentiate into a particular cell type, they may provide an almostunlimited source of cells for transplantation for the treatment of manydevastating degenerative diseases such as diabetes, Parkinson's diseaseand Alzheimer's disease (Biswas et al., 2007; Kim et al., 2007;Zimmermann et al., 2007).

Only recently, it has been shown that somatic cells may be geneticallymodified to redifferentiate into a state that is in terms of pheno- andgenotype as well as pluripotency similar to ES cells (Takahashi andYamanaka, 2006; Okita et al., 2007; Wernig et al., 2007). The so-called“reprogramming” of somatic cells is a valuable tool to understand themechanisms of regaining pluripotency and further opens up thepossibility to generate patient-specific pluripotent stem cells.Reprogramming of mouse and human somatic cells into pluripotent stemcells, designated as induced pluripotent stem (iPS) cells, has beenpossible with the expression of the transcription factor quartet Oct4,Sox2, c-Myc, and Klf4.

Presently, although it is widely acknowledged that iPS cells have agreat potential for medical applications such as, e.g., patient-specificregenerative cell therapy, the currently employed methods to generateiPS cells prevent their use in the medical field. Specifically, theretroviral vectors used to introduce and express the combination ofseveral reprogramming factors randomly integrate into the genome inmultiple copies, preferably into the vicinity or into active endogenousgenes and hence may cause activating or inactivating mutations of canceror tumor suppressor genes, respectively. Thus, the generation of iPScells using a method that minimizes the degree of modification of thetarget cell's genome may boost the clinically safe application of thisapproach.

Accordingly, the present invention relates in a first embodiment to amethod of generating an induced pluripotent stem (iPS) cell comprisingthe step of introducing into a target cell one or two coding sequenceseach giving rise upon transcription to a factor that contributes to thereprogramming of said target cell into an induced pluripotent stem celland selected from Oct3/4 or a factor belonging to the Myc, Klf and Soxfamilies of factors, wherein the target cell endogenously expresses atleast the factors that are not encoded by the coding sequences to beintroduced and selected from Oct3/4 or factors belonging to the Myc, Klfand Sox families of factors, and wherein the cell resulting from theintroduction of the one or two coding sequences expresses thecombination of factor Oct3/4 and at least one factor of each family offactors selected from the group of Myc, Klf and Sox.

An “induced pluripotent stem (iPS) cell” is a cell that exhibitscharacteristics similar to embryonic stem cells (ESCs). Saidcharacteristics include, for example, unlimited self renewal in vitro, anormal karyotype, a characteristic gene expression pattern includingstem cell marker genes like Oct3/4, Sox2, Nanog, alkaline phosphatase(ALP) and stem cell-specific antigen 3 and 4 (SSEA3/4), and the capacityto differentiate into specialized cell types (Hanna, J., et al. (2007).Science 318(5858): 1920-3; Meissner, A., et al. (2007). Nat Biotechnol25(10): 1177-81; Nakagawa, M., et al. (2007). Nat Biotechnol.; Okita,K., et al. (2007). Nature 448(7151): 313-7; Takahashi, K., et al.(2007Cell 131(5): 861-72; Wernig, M., et al. (2007). Nature 448(7151):318-24; Yu, J., et al. (2007). Science 318(5858): 1917-20; Park, I. H.,et al. (2008). Nature 451(7175): 141-6). The state of the art generationof iPS cells from fibroblast cultures has been described in Takahashi,Okita, Nakagawa, Yamanaka (2007) Nature Protocols 2(12). Thepluripotency of murine iPS cells can tested, e.g., by in vitrodifferentiation into neural, glia and cardiac cells and the productionof germline chimaeric mice through blastocyst injection. Human iPS cellslines can be analyzed through in vitro differentiation into neural, gliaand cardiac cells and their in vivo differentiation capacity can betested by injection into immunodeficient SCID mice and thecharacterisation of resulting tumors as teratomas.

iPS cells can generally be evaluated and classified according to thefollowing cellular biological properties:

Morphology: iPS cells are morphologically similar to embryonic stemcells (ESCs). Each cell has a round shape, large nucleolus and scantcytoplasm. Colonies of iPS cells are also similar to that of ESCs. HumaniPS cells form sharp-edged, flat, tightly-packed colonies similar tohESCs whereas mouse iPS cells form the colonies similar to mESCs, lessflatter and more aggregated colonies than that of hESCs.

Growth properties: Doubling time and mitotic activity are cornerstonesof ESCs, as stem cells must self-renew as part of their definition. iPScells are mitotically active, actively self-renewing, proliferating, anddividing at a rate equal to ESCs.

Stem cell markers: iPS cells express cell surface antigenic markersexpressed on ESCs. Human iPSCs express the markers specific to hESC,including SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49/6E, and Nanog.Mouse iPS cells express SSEA-1 but not SSEA-3 nor SSEA-4, similarly tomESCs.

Stem Cell Genes: iPS cells express genes expressed in undifferentiatedESCs, including, e.g., Oct3/4, Sox2, Nanog, GDF3, REX1, FGF4, ESG1,DPPA2, DPPA4, and hTERT.

Telomerase activity: Telomerases are necessary to sustain cell divisionunrestricted by the Hayflick limit of ˜50 cell divisions. hESCs expresshigh telomerase activity to sustain self-renewal and proliferation, andiPS cells also demonstrate high telomerase activity and express hTERT(human telomerase reverse transcriptase), a necessary component in thetelomerase protein complex.

Pluripotency: iPS cells are capable of differentiation in a fashionsimilar to ESCs into fully differentiated tissues. For example, iPScells injected into immunodeficient mice spontaneously form teratomasafter nine weeks. Teratomas are tumors of multiple lineages containingtissue derived from the three germ layers endoderm, mesoderm andectoderm; this is unlike other tumors, which typically are of only onecell type. Teratoma formation is a landmark test for pluripotency.Further, hESCs in culture spontaneously form ball-like embryo-likestructures termed “embryoid bodies”, which consist of a core ofmitotically active and differentiating hESCs and a periphery of fullydifferentiated cells from all three germ layers. iPS cells also formembryoid bodies and have peripheral differentiated cells. BlastocystInjection: hESCs naturally reside within the inner cell mass(embryoblast) of blastocysts, and in the embryoblast, differentiate intothe embryo while the blastocyst's shell (trophoblast) differentiatesinto extraembryonic tissues. The hollow trophoblast is unable to form aliving embryo, and thus it is necessary for the embryonic stem cellswithin the embryoblast to differentiate and form the embryo. iPS cellscan be injected by micropipette into a trophoblast, and the blastocystis transferred to recipient females. Chimeric living mouse pups can thusbe created, i.e. mice with iPS cell derivatives incorporated all acrosstheir bodies with a varying degree of chimerism.

Promoter demethylation: Methylation is the transfer of a methyl group toa DNA base, typically the transfer of a methyl group to a cytosinemolecule in a CpG site (adjacent cytosine/guanine sequence). Widespreadmethylation of a gene interferes with expression by preventing theactivity of expression proteins or recruiting enzymes that interferewith expression. Thus, methylation of a gene effectively silences it bypreventing transcription. Promoters of pluripotency-associated genes,including for example Oct3/4, Rex1, and Nanog, are demethylated in iPScells, demonstrating their promoter activity and the active promotionand expression of pluripotency-associated genes in iPSCs.

Histone demethylation: Histones are compacting proteins that arestructurally localized to DNA sequences that can effect their activitythrough various chromatin-related modifications. H3 histones associatedwith, e.g., Oct3/4, Sox2, and Nanog are demethylated, indicating theexpression of Oct3/4, Sox2, and Nanog.

The term “introducing” as used in accordance with the present inventionrelates to the process of bringing the coding sequences into the targetcell and subsequently incorporation of said coding sequences into thegenomic DNA of the target cell. This process is generally known asstable transfection and methods for stable transfection are well-knownto the person skilled in the art and described, e.g., in Bonetta,L.,(2005), Nature Methods 2, 875-883. Due to the low rate ofreprogramming events taking place in transfected cells it isadvantageous to rely on an efficient stable transfection method. Hence,the coding sequences are preferably introduced into a target cell by amethod achieving high transfection/infection efficiency. For example,transfection/infection efficiencies of at least 30%, at least 50%, or atleast 80% are preferred. Suitable methods include, for example,lipofection, electroporation, nucleofection, magnetofection or viralvector infection. Preferably, retroviral vectors are used to achievestable transfection of the target cells as said vectors not only mediateefficient entry of the coding sequences into the target cell but alsotheir integration into the genomic DNA of the target cell. Retroviralvectors have shown to be able to transduce a wide range of cell typesfrom different animal species, to integrate genetic material carried bythe vector into target cells, to express the transduced coding sequencesat high levels, and, advantageously, retroviral vectors do not spread orproduce viral proteins after infection. Suitable retroviral vectorsystems are well-known to the person skilled in the art such as, e.g.,retroviral vectors with the MoMuLV LTR, the MESV LTR, lentiviral vectorswith various internal promoters like the CMV promoter, preferably withenhancer/promoter combinations that show silencing of transgeneexpression in embryonic/pluripotent cells. Episomal vector systems likeadenovirus vectors, other non-integrating vectors, episomallyreplicating plasmids could also be used. Preferably, the retroviral MXvector system is used in the method of the invention (Kitamura et al.,(2003), Exp Hematol., 31(11):1007-1014).

Target cells to be used in the method of the invention can be derivedfrom existing cells lines or obtained by various methods including, forexample, obtaining tissue samples in order to establish a primary cellline. Methods to obtain samples from various tissues and methods toestablish primary cell lines are well-known in the art (see e.g. Jonesand Wise, Methods Mol Biol. 1997). Suitable somatic cell lines may alsobe purchased from a number of suppliers such as, for example, theAmerican tissue culture collection (ATCC), the German Collection ofMicroorganisms and Cell Cultures (DSMZ) or PromoCell GmbH, Sickingenstr.63/65, D-69126 Heidelberg. In accordance with the method of theinvention, a suitable target cell endogenously expresses factorsselected from Oct3/4 or factors belonging to the Myc, Klf and Soxfamilies of factors, wherein said factors in combination withexogenously introduced factors selected from the complementary set offactors, i.e. Oct3/4 or factors belonging to the Myc, Klf and Soxfamilies of factors, are capable to reprogram a non-pluripotent targetcell into an iPS cell. The cell resulting from the introduction of theone or two coding sequences expresses the combination of factor Oct3/4and at least one factor of each family of factors selected from thegroup of Myc, Klf and Sox. The person skilled in the art is well-awareof methods to determine whether at least two of the above-describedfactors are endogenously expressed in a target cell. Such methodsinclude, e.g., western blotting, realtime-PCR or intercellularstainings. The skilled person is further capable to realize withoutfurther ado which exogenous factor(s) are needed to complement the setof endogenously expressed factors in order to generate a cell thatexpresses the combination of Oct3/4 and at least one factor of eachfamily of factors selected from the group of Myc, Klf and Sox toinitiate reprogramming of the target cell into an iPS cell. The cellinto which the coding sequence(s) in expressible form have beenintroduced thus expresses a set of factors consisting of Oct3/4 and atleast one factor of each family of factors selected from the group ofMyc, Klf and Sox.

The invention also encompasses embodiments where a coding sequence isintroduced that is already endogenously present in the target cell. Thismay be effected, e.g., in cases where the endogenous coding sequence isexpressed only at a low level with the effect that the correspondingfactor does not or not sufficiently contribute to the reprogramming ofthe target cell.

The term “coding sequence” relates to a nucleotide sequence that upontranscription gives rise to the encoded product. The transcription ofthe coding sequence in accordance with the present invention can readilybe effected in connection with a suitable promoter. Preferably, thecoding sequence corresponds to the cDNA sequence of a gene that givesrise upon transcription to a factor that contributes to thereprogramming of a target cell into an induced pluripotent stem cell,wherein the reprogramming factors in accordance with the method of theinvention are selected from Oct3/4 or factors belonging to the Myc, Klfand Sox families of factors.

A “factor that contributes to the reprogramming of a target cell into aninduced pluripotent stem cell” relates to a factor that is capable ofcontributing to the induction of the reprogramming of target cells intoinduced pluripotent stem cells, wherein the factor is selected fromOct3/4 and factors belonging to the Myc, Klf and Sox families offactors. Such reprogramming factors include, for example, Oct3/4, Sox2,Sox1, Sox3, c-Myc, n-Myc, I-Myc, Klf1, Klf2, Klf4, Klf5, and the like,or mutants thereof with retained reprogramming capabilities. Saidcontribution to the reprogramming may be in the form of, for example,changing the methylation pattern of a cell to one similar to anembryonic stem cell, shifting the expression profile of a cell towardsthe expression profile of an embryonic stem cell or affectingconformation of the aggregated nuclear DNA by modulating the histonebinding similar to that observed in an embryonic stem cell wherein eachof said changes may be effected either alone or in combination by asuitable reprogramming factor. Apart from the above-recited factors, theskilled person is aware of methods to identify further suitablereprogramming factors such as, e.g., bisulphite genomic sequencing,RT-PCR, real-time PCR, microarray analysis, karyotype analysis, teratomaformation, alkaline phosphatase staining, all of which are well-known tothe person skilled in the art and are, for example described in Okita,K., et al. (2007), Nature 448(7151): 313-7; Park, I. H., et al. (2008),Nature 451(7175): 141-6; Takahashi, K., et al. (2007), Cell 131(5):861-72; Wernig, M., et al. (2007), Nature 448(7151): 318-24; Takahashi,K. et al. (2007), Nat Protoc 2(12): 3081-9; or Hogan, B., et al. (1994),“Manipulating the Mouse Embryo: A Laboratory Manual”, Cold SpringHarbour Press.

Oct3/4 belongs to the family of octamer (“Oct”) transcription factors,and plays a role in maintaining pluripotency. The absence of Oct3/4 incells normally expressing Oct3/4, such as blastomeres and embryonic stemcells, leads to spontaneous trophoblast differentiation. Thus, thepresence of Oct3/4 contributes to the pluripotency and differentiationpotential of embryonic stem cells. Various other genes in the “Oct”family, including Oct1 and Oct6, fail to elicit induction, thusdemonstrating the exclusiveness of Oct3/4 to the induction process. Theterm “Oct4” is used herein interchangeably with the term “Oct3/4”.

The Sox family of genes is associated with maintaining pluripotencysimilar to Oct3/4, although it is associated with multipotent andunipotent stem cells in contrast to Oct3/4, which is exclusivelyexpressed in pluripotent stem cells. Klf4 of the Klf family of genes wasinitially identified as a factor for the generation of mouse iPS cellsand was demonstrated as a factor for generation of human iPS cells.

The genes belonging to the Myc family are proto-oncogenes implicated incancer. It was demonstrated that c-Myc is a factor implicated in thegeneration of mouse iPS cells and that it was also a factor implicatedin the generation of human iPS cells. Introduction of the “Myc” familyof genes into target cells for the generation of iPS cells is troublingfor the eventuality of iPS cells as clinical therapies, as 25% of micetransplanted with c-Myc-induced iPS cells developed lethal teratomas.N-Myc and I-Myc have been identified to replace c-myc with similarefficiency.

The term “reprogramming” as used in accordance with the presentinvention relates to the process of changing the geno- and phenotypicalprofile of a cell that results in a cell that is geno- and/orphenotypically similar to an embryonic stem cell. Said changes comprise,for example, changes in the methylation pattern, shifts in theexpression profile or conformational changes of the aggregated nuclearDNA as described herein above.

The above applies mutatis mutandis to other embodiments described hereinbelow.

The method of the invention is based upon the surprising finding that itis possible to obtain iPS cells by the introduction of only tworeprogramming factors. Prior to this finding the dogma of the prior artwas that viable iPS cells which are functional in in vivo experiments,i.e. capable of contributing to the three germlayers, could onlysuccessfully be generated by introducing at least three, but moreeffectively by introducing a combination of four reprogramming factors.

Exemplarily, it was demonstrated that murine neural stem cells (NSCs)could be reprogrammed by introducing a combination of four (4F), three(3F) and only two (2F) reprogramming factors as well as only onereprogramming factor using the retroviral MX vector system. The NSCswere established from adult OG2/Rosa26 heterozygous transgenic micebrain (Ryan, A. K. & Rosenfeld, M. G., Genes Dev 11, 1207-25 (1997); Do,J. T. & Scholer, H. R., Stem Cells 22, 941-9 (2004); Pollard, S. M.,Conti, L., Sun, Y., Goffredo, D. & Smith, A., Cereb Cortex 16 Suppl 1,i112-20 (2006)), expressing GFP under the control of the Oct4 promoter(Oct4-GFP) and the lacZ transgene from the constitutive Rosa26 locus.

First observed were GFP+ colonies in NSC cultures infected with Oct4 andKlf4 (2F OK) and 1-2 weeks later in those infected with Oct4 and c-Myc(2F OM) (Table 1).

TABLE 1 Overview of the applied combinations of reprogramming factors,timing of GFP colony formation, and establishment of iPS cell linesTiming of GFP-positive Establishment of iPS cell Transfected factorscolonies line OK 2-3 weeks + OM 3-4 weeks +

The 2F OM iPS cells were further analyzed and showed an ESC-likeexpression pattern as well as contributing to the three germ layers interatomas.

2F OK iPS cells were compared with 4F (generated using standard approachof introducing 4 reprogramming factors to generate iPS cells) iPS cellsand ESCs. On day 14 post-infection, 5 GFP+ colonies were dissociated andpropagated under ESC culture conditions (FIG. 1c, f ), yielding 3 (i.e.60%) 2F OK iPS cell clones (B-2, D-7 and F-4) that were morphologicallyindistinguishable from ESCs (FIG. 1d, g ). No colonies had formed fromNSCs infected with control virus (MX) (FIG. 1e, h ). The reprogrammingefficiencies were estimated from the number of Oct4-GFP+ colonies andtransduction rates with MX-GFP control virus on NSCs for the 2F OK iPSand 4F iPS by time course (FIG. 1i, j ). Thereby a reprogrammingefficiency of 3.6% for 4F reprogramming of NSCs and 0.11% for the twofactors approach was calculated, what is comparable to reprogramming offibroblasts with selection (below 0.08%, Takahashi, K. & Yamanaka, S.,Cell 126, 663-76 (2006); Okita, K., Ichisaka, T. & Yamanaka, S., Nature448, 313-7 (2007); Wernig, M. et al., Nature 448, 318-24 (2007)) andwithout selection (0.5%; Meissner, A., Wernig, M. & Jaenisch, R., NatBiotechnol 25, 1177-81 (2007)) (FIG. 1j ). Transduction with all 4factors had a positive impact on the timing and number of GFP+ colonies.Integration of the viral transgenes was confirmed by genotyping PCR. Theviral transgenes of all 4 factors were detected in 4F iPS cells, while2F OK iPS cells only contained the Oct4 and Klf4 transgenes.

2F OK iPS cells stained positive for SSEA-1 and alkaline phosphatase,and exhibited ES cell marker genes expression patterns similar to 4F iPScells and ESCs (FIG. 2a ). qRT-PCR results demonstrated that expressionof endogenous Oct4, Sox2, c-Myc, and Klf4 in 2F OK iPS cells wascomparable to ESCs, and the silencing of the viral transcripts in 2F OKiPS cells with a 1000-fold reduction after 30 days. 2F iPS global geneexpression also clusters close to ESCs and 4F iPS (FIG. 2b ). Scatterplots of DNA microarray analyses demonstrated a higher similaritybetween 2F iPS cells and ESCs than between 2F iPS cells and NSCs (FIG.2c, d ). Thus, 2F iPS cells (clone F-4) seemed to be very similar tomouse ESCs at the global transcription level.

The differentiation ability of 2F OK iPS cells was confirmed by in vitrodifferentiation into embryoid bodies (EBs). These cells expressed theectoderm (Tuj1), endoderm (α-fetoprotein), and mesoderm marker Flk1(expressed by beating cells mimicking cardiomyocytes) (FIG. 3a ).Teratomas contained derivatives of all three germ layers (FIG. 3b ), andexpressed markers of the three germ layer. No teratoma had formed fromdonor cells (NSCs). These data demonstrate that 2F OK iPS cells exhibita pluripotent phenotype in in vitro and in vivo.

To investigate their developmental potential, 2F OK iPS cells wereaggregated with 8-cell-stage embryos. iPS cells had contributed to theformation of the inner cell mass in developing blastocysts (FIG. 4a ).After transferring aggregated blastocysts into pseudopregnant females,16 live embryos were obtained on E13.5, of which 2 embryos showed germcell contribution in the foetal gonads, judged from Oct4-GFP expression(FIG. 4b ). X-gal staining (visualising the NSC donor cells that carrythe Rosa β-geo26 (lacZ) transgene) of embryonic tissue from wholeembryos revealed that in the resulting chimeras, 2F OK iPS cellscontributed to the development of all three germ layers (FIG. 4c, e ).The strictest test for developmental potency tetraploid (4N) embryoaggregation (n=122) resulted in 2 dead (arrested) embryos at E13.5 (FIG.4d ). This is within the normal rate for 4N embryo aggregation and wasnot related to deficient pluripotency of the introduced cells. Thesedata demonstrate that iPS cells can give rise to all of the tissues of alate-stage embryo. In diploid (2N) aggregation, PCR genotyping showedthat 2 out of 13 chimeras were positive for the Oct4-GFP allele of thedonor cell (FIGS. 4f and g (top panel)). To assess whether 2F OK iPScells can contribute to the germline, chimeras were mated with CD-1females. Two out of 12 pups had a Oct4-GFP allele and 1 out of 12 micehad a lacZ allele. Since the donor cells are derived from aheterogeneous mouse (Oct4+/− Rosa26+/−), they also have the Oct4 andKlf4 transgenes (FIG. 4g (bottom panel)). No tumour formation wasobserved from adult chimeras and F1 mice by the age of 17 weeks and 3weeks respectively. This finding indicates that 2F OK iPS cells cancontribute the full term development of chimera, resulting in a nextgeneration (F1) of viable pups and thus suggests that the iPS cells havea similar developmental property like ESCs.

As described in detail in Example 9 below, the inventors were able toalso demonstrate conversion of human cells into pluripotent stem cellsby the introduction of two or only one reprogramming factor. Saidreprogramming factors were Oct4 or Oct4 and Klf4.

In conclusion, the above findings demonstrate the successful generationof iPS cells using two reprogramming factors or only one reprogrammingfactor. The advantage of the method of the invention lies in the use ofonly two or even only one retroviral vector for stable transfection ofone or two reprogramming factors. The possibility of inducing iPS cellswith a reduced number of retroviral vectors as compared to prior artapproaches presents a major step towards the minimization of geneticmodulation of the initial cell population to be reprogrammed.Accordingly, the risk of formation of aberrant and tumourigenic cells issignificantly decreased, hence allowing the generation of iPS cellssuitable for therapeutic purposes, inter alia.

In a preferred embodiment of the method of the invention, the factorsbelonging to the factor families of Myc, Klf and Sox and endogenouslyexpressed by or encoded by the coding sequences to be introduced intothe target cell are selected from the group consisting of I-Myc, n-Myc,c-Myc, Klf1, Klf2, Klf4, Klf15, Sox1, Sox2, Sox3, Sox15 and Sox18.

The coding sequence of, for example, murine Oct3/4, Sox2, c-Myc, andKlf4 can be found in SEQ ID NOs: 1, 5, 9 and 13, respectively. Theprotein sequence of murine Oct3/4, Sox2, c-Myc and Klf4 can be found inSEQ ID NOs: 2, 6, 10 and 14, respectively. The coding sequence of humanOct3/4, Sox2, c-Myc and Klf4 can be found in SEQ ID NOs: 3, 7, 11 and15, respectively. The protein sequence of human Oct3/4, Sox2, c-Myc andKlf4 can be found in SEQ ID NOs: 4, 8, 12 and 16, respectively. Theskilled person is in the position to determine the coding sequences ofreprogramming factors for any target species using methods well-known inthe art. For example, he can retrieve data relating to sequence andfunction from databases such as, for example, the databases maintainedby the National Center for Biotechnology Information (NCBI) andaccessible via the World Wide Web under http://www.ncbi.nlm.nih.gov/.Further, databases for comparative genomics include without limitation,a database maintained also by the NCBI at http://www.dcode.org/, adatabase for protein annotations for all completely sequenced organismsaccessible at http://supfam.org/SUPERFAMILY/, a database comprisinggenome information for various species accessible athttp://www.cbs.dtu.dk/services/GenomeAtlas/, or a database comprisinggene clusters accessible at http://phigs.jgi-psf.org/. Said databasesallow the skilled person to identify coding sequences for reprogrammingfactors in other species starting from the sequences known for mice andhumans by, for example, performing cross-species sequence alignments toidentify homologous genes.

Several, only recently published scientific articles (Hanna, J., et al.(2007). Science 318(5858): 1920-3; Meissner, A., et al. (2007). NatBiotechnol 25(10): 1177-81; Nakagawa, M., et al. (2007). NatBiotechnol.; Okita, K., et al. (2007), Nature 448(7151): 313-7;Takahashi, K., et al. (2007), Cell 131(5): 861-72; Wernig, M., et al.(2007). Nature 448(7151): 318-24; Yu, J., et al. (2007). Science318(5858): 1917-20; Park, I. H., et al. (2008). Nature 451(7175): 141-6)have shown that transcription factors belonging to the Oct, Sox, Klf andMyc families are capable of contributing to the induction ofreprogramming in murine as well as human somatic cells.

In another preferred embodiment, the target cell does not endogenouslyexpress one of the factors encoded by the one or two coding sequences tobe introduced into said target cell.

Methods of assessing endogenous expression of factors are well-known tothe skilled person and described elsewhere in this specification. Inorder to generate iPS cells in accordance with the method of theinvention the target cell may not endogenously express one of thefactors encoded by the one or two coding sequences that are to beintroduced into the target cell. For example, it could be demonstratedthat Oct3/4 was not expressed in murine neural stem cells as targetcells, whereas Sox2, Klf4 and c-Myc were endogenously expressed.Exogenous introduction of Oct3/4 and subsequent expression wassufficient to complement the quartet of reprogramming factors and inducegeneration of iPS cells.

In another preferred embodiment, the target cell is a multipotent stemcell.

Multipotent stem cells can give rise to several other cell types, butthose types are limited in number. This is in stark contrast topluripotent stem cells being capable of differentiating into any celltype. An example of a multipotent stem cell is a hematopoietic cell,found e.g. in bone marrow, cord blood or circulation, that can developinto several types of blood cells, but cannot develop into other typesof cells. Another example of multipotent cells are neural stem cells.Multipotent cells are particularly suitable as reprogramming targetcells, since they already have reprogramming factors upregulated.

In more preferred embodiment, the multipotent stem cell is an ectodermalcell.

The ectoderm is the outermost of the three primary germ cell layers (theother two being the mesoderm and endoderm) that make up the very earlyembryo. It differentiates to give rise to many important tissues andstructures including the outer layer of the skin and its appendages (thesweat glands, hair, and nails), the teeth, the lens of the eye, parts ofthe inner ear, neural tissue, brain, and spinal cord. Ectodermal cellsas multipotent stem cells are particularly suitable as target cells,since ectodermal cells like neural stem cells already endogenouslyexpress reprogramming factors.

In another preferred embodiment, the target cell is a neural stem cell(NSC).

Neural stem cells exist not only in the developing mammalian nervoussystem but also in the adult nervous system of all mammalian organisms,including humans. Neural stem cells can also be derived from moreprimitive embryonic stem cells. The location of the adult stem cells andthe brain regions to which their progeny migrate in order todifferentiate remain unresolved, although the number of viable locationsis limited in the adult (for a review see Gage, 2000). Neural stem cellsare particularly suitable as target cells as they already endogenouslyexpress reprogramming factors.

In a more preferred embodiment, the coding sequence to be introducedencodes the factor Oct3/4.

As outlined herein above and demonstrated in the Examples below, theintroduction of Oct3/4 alone into a neural stem cell was sufficient togenerate iPS cells. As c-Myc increases tumourigenicity in chimera pups(Okita, K., Ichisaka, T. & Yamanaka, S., Nature 448, 313-7 (2007)), therecent studies demonstrating iPS cell generation without the c-Mycretroviral integration (Nakagawa, M. et al., Nat Biotechnol 26, 101-106(2008); Wernig, M., Meissner, A., Cassady, J. P. & Jaenisch, R., CellStem Cells 2, 11-12 (2008)) present a significant improvement. However,the possibility of inducing iPS cells without c-Myc as presented in thisembodiment in combination with the reduced number of retroviral vectorsis a major step towards the minimization of genetic modulation of theinitial cell population to be reprogrammed.

The same target cell could also be reprogrammed by the introduction ofonly two factors. Accordingly, in a different more preferred embodiment,the two coding sequences to be introduced encode factors Oct3/4 andc-Myc or Oct3/4 and Klf4.

In an even more preferred embodiment, the target cell endogenouslyexpresses the factors c-Myc, Klf4 and Sox2.

It could be shown that the target cell when endogenously expressing theabove combination of reprogramming factors was amenable to reprogrammingupon introduction of one or two exogenous reprogramming factors, such asOct3/4 alone or Oct3/4 and c-Myc or Oct3/4 and Klf4.

In an even more preferred embodiment, the target cell endogenouslyexpresses the factors c-Myc, Klf4 and Sox2 at levels at least 10-foldlower or at most 10-fold higher as compared to the correspondingexpression levels in embryonic stem cells of the same genus as thetarget cell.

It is advantageous in accordance with the method of the invention whenthe expression levels of the endogenous reprogramming factors are in acertain range as compared to the expression levels in ESCs of the samegenus as the target cell. Preferably, the target cell endogenouslyexpresses the reprogramming factors c-Myc, Klf4 and Sox2 at levels atleast 10-fold lower or at most 10-fold higher as compared to thecorresponding expression levels of said factors in ESCs. More preferredis the expression of Sox2 about two-fold higher, c-Myc about 10-foldhigher and/or Klf4 about 8-fold lower than in ESCs belonging to the samegenus as the target cells. The term “about” as used in the context ofthe present invention refers to an average deviation of maximum +/−20%,preferably +/−10%. Also envisaged is the expression at levels at least8-, 6-, 5-, 4-, 3- or 2-fold lower or at most 8-, 6-, 5-, 4-, 3- or2-fold higher or any arbitrary number in-between as compared to saidESCs.

In a more preferred embodiment, the target cell is a murine or a humanneural stem cell.

Furthermore, the invention relates to an induced pluripotent stem cellgenerated by the method of the invention.

Pluripotent stem cells generated by the method of the invention may beuseful in a variety of experimental as well as therapeutic settings. Forexample, the use of the iPS cells, of cells derived therefrom bydifferentiation or tissues generated from said iPS cells or cellsderived therefrom as a therapeuticum or diagnosticum, within gene orcell transplantation treatments, for the identification and validationof genomic targets as well as Drug screening approaches are envisaged.

The culture conditions for iPS cells are the same as established forembryonic stem cells of the corresponding species and are well-known tothe person skilled in the art. Generally, cell culture methods, such as,for example, media constituents, marker choice and selection, cellquantification and isolation, are methods well-known in the art anddescribed, for example, in “Practical Cell Culture Techniques”, Boultonet Baker (eds), Humana Press (1992), ISBN 0896032140; “Human CellCulture Protocols”, Gareth E. Jones, Humana Press (1996), ISBN089603335X and exemplarily in the example section. Methods for culturingand maintaining cells in culture are well-known in the art; growth mediaand other cell culture related material as well as instructions andmethods for successful culturing of cells can, for example, be obtainedat Sigma-Aldrich or Invitrogen.

Further, the invention relates to a method of identifying a compoundthat contributes to the reprogramming of a target cell into an inducedpluripotent stem cell comprising the steps of: (a) reprogramming atarget cell according to the method of the invention, wherein one codingsequence to be introduced is replaced by the compound to be tested; and(b) assessing whether iPS cells are formed in the presence and absenceof the compound to be tested, wherein the formation of iPS cells fromtarget cells in which the compound to be tested has been introduced isindicative of the compound contributing to the reprogramming of a targetcell into an induced pluripotent stem cell.

In accordance with the invention the compound to be tested may be one ormore nucleic acids, such as DNA, cDNA, RNA, dsRNA, sRNA, shRNA, miRNA,proteins, peptides, small molecules (organic or inorganic), chemicals orany combination thereof.

Reprogramming a target cell in accordance with the method of theinvention has been described herein-above. Depending on the nature ofthe compound to be tested the method of the invention may need to bemodified as regards the introduction step of the compound into thetarget cell. For example, if other transcription factors are to beevaluated the corresponding coding sequences may be introduced asdescribed above without modification. In contrast, chemicals or smallmolecules may be introduced by exogenously adding the respectivecompound to the cell medium and taking advantage of passive or activecellular uptake mechanisms. The skilled person is well-aware of methodsthat allow the introduction of any compound to be tested into the cell,preferably into the nucleus, in order to test whether the compound canindeed substitute the factor it replaces and accordingly inducereprogramming of the target cell. Nucleic acids, such as DNA, cDNA, RNA,dsRNA, sRNA, shRNA, miRNA can be introduced by transfection orinfection, small molecules (organic or inorganic), chemicals just bepenetration throughout the membrane.

The skilled person is well aware of methods to assess whether iPS cellsare formed in the presence and absence of the compound to be tested.Criteria for the classification of an iPS cell are known to the skilledperson and have been described herein above. Depending on the criteriato be assessed the methods vary and may include, e.g., visual control bymicroscopy, expression analysis of markers, teratoma formation alone orin combination.

The finding of the invention that cells endogenously expressing a set offactors contributing to the reprogramming of said cell may becomplemented by the exogenous addition of further factors resulting in acell expressing a quartet of reprogramming factors, i.e. Oct3/4 and afactor of each family of factors Myc, Klf and Sox, leading to theinduction of reprogramming of the target cell, significantly simplifiesthe identification of compounds that can replace a factor in thereprogramming process. As only one or two factors have to be introducedinstead of three or the entire set of four factors known in the art togenerate cells suitable for screening, a considerable reduction of time,costs and experimental difficulties is achieved. Also high throughputscreening approaches for novel reprogramming factors will evidently beimproved as regards time and efficiency with a reduced set of factorsnecessary to be introduced.

Also, the invention relates to a method of generating a transgenicnon-human animal comprising the steps of: (a) introducing the inducedpluripotent stem cell of the invention or generated by the method of theinvention into a non-human preimplantation embryo; (b) transferring theembryo of step (a) into the uterus of a female non-human animal; and (c)allowing the embryo to develop and to be born.

The term “transgenic non-human animal” as used in accordance with theinvention relates to an animal in which there has been effected adeliberate modification of its genome by methods described herein.

The method of the invention of generating a transgenic non-human animalis preferably carried out according to methods that have beenestablished for generating transgenic non-human animals by the use ofembryonic stem cells, however, replacing the embryonic stem cells withiPS cells of the invention. Said methods are well-known in the art(Hogan, B., R. Beddington, et al. (1994), “Manipulating the MouseEmbryo: A Laboratory Manual”, Cold Spring Harbour Press; Hanna, J., etal. (2007), Science 318(5858): 1920-3; Meissner, A., et al. (2007), NatBiotechnol 25(10): 1177-81; Nakagawa, M., et al. (2007), NatBiotechnol.; Okita, K., et al. (2007), Nature 448(7151): 313-7;Takahashi, K., et al. (2007), Cell 131(5): 861-72; Wernig, M., et al.(2007), Nature 448(7151): 318-24; Yu, J., et al. (2007), Science318(5858): 1917-20; Park, I. H., et al. (2008), Nature 451(7175):141-6). In brief, introduction of the iPS cell into a non-humanpreimplantation embryo, like a morula or a blastocyst, is preferablyeffected by microinjection into a morula or blastocyst or by aggregationof iPS cells with 8-cell or morula embryos. Said chimaeric embryo isthen transferred into the uterus of a pseudopregnant non-human femalewhere it develops into an embryo that is finally born (cf. Example 8).

Generating a transgenic non-human animal line from iPS cells is based onthe pluripotence of said iPS cells (i. e., their ability, once injectedinto a host developing embryo, such as a blastocyst or morula, toparticipate in embryogenesis and to contribute to the germ cells of theresulting animal). As outlined above, the blastocysts containing theinjected iPS cells are allowed to develop in the uteri of pseudopregnantnon-human females and are born as chimeras. The resultant transgenicnon-human animals are chimeric for cells originating from iPS cells andare backcrossed to wildtype non-human animals and screened for animalscarrying only the genetic content of an iPS cell so as to identifytransgenic animals homozygous for the combination of DNA segments.

The transgenic non-human animals may, for example, be transgenic mice,rats, hamsters, dogs, monkeys, rabbits, pigs, or cows. Preferably, saidtransgenic non-human animal is a mouse.

Accordingly, the invention also relates to a transgenic non-human animalgenerated by the method of the invention.

Finally, the invention relates to a composition comprising an iPS cellgenerated by the method of the invention for gene therapy, regenerativemedicine, cell therapy or drug screening.

A composition as used herein relates to a composition that comprises iPScells and preferably further constituents that maintain cell viabilityof said cell. Such constituents are well-known to the skilled person andcomprise, for example, cell media constituents. Further, depending onthe intended application the composition may comprise additionalconstituents, for example, constituents facilitating administration to apatient.

A composition comprising the iPS cells of the invention (as well as theiPS cells of the invention per se) can be used in a variety ofexperimental as well as therapeutic scenarios. The iPS cell of theinvention having a comparatively low number of transgenic expressionelements and an overall reduced risk of developing into cancerous cellsare expected to be beneficial in gene therapy, regenerative medicine,cell therapy or drug screening.

Gene therapy, which is based on introducing therapeutic DNA constructsfor correcting a genetic defect into germ line cells by ex vivo or invivo techniques, is one of the most important applications of genetransfer. Suitable vectors and methods for in vitro or in vivo genetherapy are described in the literature and are known to the personskilled in the art (Davis P B, Cooper M J., AAPS J. (2007), 19;9(1):E11-7; Li S, Ma Z., Curr Gene Ther. (2001), 1(2):201-26). Inaccordance with the invention, cells obtained from a patient could, forexample, be genetically corrected by methods known in the art andsubsequently be reprogrammed into iPS cells having the pheno- andgenotype of ES cells, by the method of the invention. This evidences theapplicability of iPS cells in gene therapy and/or cell therapy.Regenerative medicine can be used to potentially cure any disease thatresults from malfunctioning, damaged or failing tissue by eitherregenerating the damaged tissues in vivo or by growing the tissues andorgans in vitro and subsequently implanting them into the patient. TheiPS cells of the invention being capable of differentiating intovirtually any tissue (ectoderm, mesoderm, endoderm cells) can be used inany aspect of regenerative medicine and hence drastically reduce theneed for ES cells.

The iPS cells of the invention can also be used to identify drug targetsand test potential therapeutics hence reducing the need for ES cells andin vivo studies. Experimental setups and methods to identify and/orassess effects of a potential drug including, for example, target-siteand -specificity, toxicity, bioavailability, are well-known to theperson skilled in the art.

Further, the iPS cells may be used to study the prevention and treatmentof birth defects or study cell differentiation.

Also, the iPS cells of the invention may be useful in an experimentalsetting—besides therapeutic applications—to study a variety of aspectsrelated to dedifferentiation when inducing reprogramming of a targetcell such as, e.g., spatiotemporal shifts in the expression pattern ofgenes or of methylation patterns, or the morphological changes leadingto changes in aggregation behaviour. The iPS cells can further besubject to studies relating to, e.g., gene therapy, gene targeting,differentiation studies, tests for safety and efficacy of drugs,transplantation of autologous or allogeneic regenerated tissue, tissuerepair (e.g., nervous system, heart muscle), diseases like, e.g.,Parkinson's disease, heart attack, diabetes, cancer, leukemia or spinalcord injury, embryonal gene expression, genetic manipulation ofembryonal genes, early embryology and fetal development, identificationof embryonic cell markers, cell migration or apoptosis.

The figures show:

FIG. 1: Generation of 2F Oct4/Klf4 (OK) iPS cells from adult NSCs ofOG2/Rosa26 transgenic mice.

a. RT-PCR and qRT-PCR analyses of Oct4, Nanog, Klf4, Sox2, and c-Myc inESCs and NSCs. β-actin was used as loading control. b. Western blotanalyses of the four factors in ESCs and NSCs.

Anti-actin antibody was used as loading control. c. Morphology of 2F OKiPS cell colony on day 14 post-infection. An ESC-like colony expressingOct4-GFP (f). d. Morphology of an established 2F OK iPS cells (cloneF-4) on day 30 post-infection, grown on irradiated MEFs. Phase contrastand Oct4-GFP (g) are shown. e. Morphology of NSCs and mock infection onday 30 post-infection (h). i. Generation of GFP-positive colonies at day7, 14, and 21 after 2F OK and 4F infection (n=3; error bars indicateds.d.). j. Reprogramming efficiency of generating 2F and 4F iPS cells(n=3). Indicated are the total numbers of GFP+ colonies per 50,000plated NSCs at day 7, 14, and 21 after infection.

FIG. 2: Gene expression profile of iPS cells.

a. RT-PCR analysis of ES cell marker gene expression in ESCs, 4F iPScells (clone A-2c), 2F OK iPS cells (clones B-2, D-7 and F-4), and NSCs.Primers are specific for transcripts from the respective endogenouslocus. β-actin was used as loading control. b. The heatmap of thedifferent expressed genes among the NSC, 2F (OK) iPS, 4F iPS and ESC.The gene hierarchical cluster was performed with a cityblock distanceand an average linkage. c. Global gene expression patterns were comparedbetween 2F iPS cells (clone F-4) and ESCs, and between 2F iPS cells(clone F-4) and NSCs with DNA microarrays. d. Black lines indicatetwo-fold changes in gene expression levels between the paired celltypes. Genes overexpressed in 2F iPS cells (clone F-4) compared withNSCs or ESCs are shown in blue; those underexpressed are shown in red.Positions of pluripotency genes Oct4, Nanog, Sox2, c-Myc, Klf4 and Lin28in scatter plots are indicated. The gene expression level is scaled inlog 2.

FIG. 3: 2F Oct4/Klf4 (OK) iPS cells (clone F-4) are pluripotent anddifferentiate in vitro and in vivo.

a. In vitro differentiation into all three germ layers. After embryoidbody formation, aggregates were transferred onto gelatine-coated platesand allowed to differentiate for another 10 days. Cells were stainedwith anti-Tuj1, anti-α-fetoprotein (AFP), or anti-Flk1. Nuclei werestained with DAPI. b. Teratomas of F-4 iPS cells containing all threegerm layers. F-4 iPS cells (1.5×10⁶ cells) were subcutaneouslyinoculated into nude mice. After 4 weeks, teratomas were stained withhaematoxylin and eosin dyes. Shown is a teratoma containing a neuralrosette (ectoderm), muscle (mesoderm), and columnar epithelium(endoderm).

FIG. 4: In vivo developmental potential of 2F Oct4/Klf4 (OK) iPS cells(clone F-4).

a. The chimeric embryos of F-4 iPS cells developed to blastocysts after24 hrs of aggregation. Fluorescence optics show Oct4-GFP cells locatedin the inner cell mass of blastocysts. b. Germline contribution of F-4iPS cells to mouse embryonic development as shown by the expression ofOct4-GFP. Embryos were analyzed with a fluorescence microscope at E13.5.c, d. The 13.5 dpc chimeric embryos (control, 2N, and 4N) were stainedwith X-gal solution. e. Histological analysis of lacZ-stained 13.5 dpcchimeric embryo (2N). f. Chimeric mouse (8-week-old) generated by F-4iPS cells. Agouti coat colour originated from F-4 iPS cells. g. PCRgenotyping of chimeras derived from F-4 iPS cell. PCR analyses wereperformed for Oct4-GFP (top panel). Germline transmission of F-4 iPScells. Genotyping of offspring from chimeric males mated with CD-1females demonstrated the presence of Oct4-GFP and lacZ allele and Oct4and Klf4 virus integrations (bottom panel). Abbreviation: Gastroint.tract.: gastrointestinal tract.

FIG. 5: One-factor hNSC-derived iPS (1F hNiPS) cell colony formation andcell line characterization.

(A) Morphology of hNSCs grown in NSC medium. (B) Colony formation ofhOCT4-infected cells 10 weeks post-infection. (C) The colony growshESC-like morphology but center of colony still remain unreprogrammedneural rosettes. (D) Typical hESC-like iPS colony growing on feederafter mechanical isolation at passage 1 (1F hNiPS clone C). (E) Highmagnification of iPS colony at passage 10. (F) 1F hNiPS colonies werestained for AP. Scale bars, 250 μm. (G) Immunocytochemical analysis ofpluripotency markers (OCT4, SSEA4, TRA-1-60 and TRA-1-81) in 2F hNiPS(clone A) and 1F hNiPS (clone C) cells. Nuclei are stained with DAPI(blue). Scale bars, 250 μm.

FIG. 6: Expression level of pluripotent markers and DNA methylationanalysis in hNSC-derived iPS (hNiPS) cells.

(A) Quantitative PCR analysis for pluripotent markers in H1 hESCs,hNSCs, 2F hNiPS clones (A, B and C) and 1F hNiPS clones (A and C). Dataare shown relative expression to H9 hESCs using primers specific forendogenous transcripts. RNA expression levels are shown on logarithmicscale. Transcripts levels were normalized to β-actin levels. Error barsindicate the s.d. from triplicates. (B) Bisulfite sequencing analysis ofOCT4 and NANOG promoter regions in H9 hESCs, hNSCs, 2F hNiPS clones (A,B and C) and 1F hNiPS clones (A and C). Each row of circles for a givenamplicon represents the methylation status of each CpG in one bacterialclone for that region. Open circles represent unmethylated CpGs, andclosed circles represent methylated CpGs. Bottom numbers of each columnindicate CpG dinucleotide locations, relative to the transcriptionalstart site (TSS; +1).

FIG. 7: In vitro differentiation of hNSC-derived iPS (hNiPS) cells intoall three germ layers.

(A) Immunofluorescence analysis shows differentiation of 2F and 1F hNiPScells into all three germ layers: endoderm (alpha-fetoprotein; AFP),mesoderm (alpha-smooth muscle actin; α-SMA) and ectoderm (β-tublin IIIb;Tuj1). Nuclei are stained with DAPI (blue). Scale bars, 100 μm. (B)Quantitative PCR analyses of one-month embryoid bodies (EBs)differentiation derived from 2F hNiPS (clone A) and 1F hNiPS (clone C)cells. Endoderm (AFP, GATA6 and Sox17), mesoderm (FOXF1 and HAND1) andectoderm (NCAM1, PAX6 and Sox1). Data are shown relative expression toeach undifferentiated parental hNiPS cells. RNA expression levels areshown on logarithmic scale. Transcripts levels were normalized toβ-actin levels.

FIG. 8: In vivo pluripotency and global gene expression profile ofhNSC-derived iPS (hNiPS) cells.

(A) Teratoma formation after transplantation of 2F hNiPS (clone A) and1F hNiPS (clone C) cells into SCID mice, and teratomas were sectionedand stained with hematoxylin and eosin at 6-8 weeks. Histologicalsection of identified cells representing all three germ layers: endoderm(respiratory epithelium; r), mesoderm (skeletal muscle; m, cartilage; c)and ectoderm (neural epithelium; n). Enlargements of sections showingrespiratory epithelium, muscle and neural epithelium indicated byarrows. Scale bars, 100 μm. (B) Heat map (left panel) and hierarchicalcluster analysis (right panel) of global gene expression from hNSCs, 1FhNiPS (clone C), 2F hNiPS (clone A) H9 hESCs and H1 hESCs (left). (C)Scatter plots comparing global gene expression profiles between 1F hNiPS(clone C) and H9 hESCs (left panel), 2F hNiPS (clone A) and H9 hESCs(middle panel), and hNSCs and 1F hNiPS (clone C) (right panel). Theblack lines indicate twofold difference in gene expression levelsbetween the paired cell populations. The transcript expression levelsare on the log² scale.

The examples illustrate the invention:

EXAMPLE 1: GENERATION OF OG2 MICE

The OG2 strain was crossed with the ROSA26 transgenic strain (Do, J. T.& Scholer, H. R., Stem Cells 22, 941-9 (2004); Szabo, P. E., Hubner, K.,Scholer, H. & Mann, J. R., Mech Dev 115, 157-60 (2002)) over severalgenerations to produce compound homozygous mice for the neo/lacZ andOct4-GFP transgenes. To derive NSCs, homozygous OG2×ROSA26 male micewere crossed with ICR females to produce heterozygous pups. Brain tissuewas collected from 5-day-old OG2×ROSA26 heterozygous mice.

EXAMPLE 2: GENERATION OF INDUCED PLURIPOTENT STEM CELLS

iPS cells and ESCs were grown on irradiated MEFs and in ESC medium (DMEMsupplemented with 15% FBS, nonessential amino acids, L-glutamine,penicillin/streptomycin, β-mercaptoethanol, and 1,000 U/ml leukemiainhibitory factor (LIF)). pMX-based retroviral vectors encoding themouse cDNAs of Oct4, Sox2, Klf4, and c-Myc were separately cotransfectedwith packaging-defective helper plasmids into 293 cells using Fugene 6transfection reagent (Roche). 48 hrs later, virus supernatants werecollected as previously described (Zaehres, H. & Daley, G. Q., (2006),Methods Enzymol 420, 49-64). NSCs derived from OG2/Rosa26 transgenicmice were seeded at a density of 5×10⁴ cells per 6-well plate andincubated with virus-containing supernatants for the four factors(1:1:1:1) or for Oct4 and Klf4 (1:1) supplemented with 6 μg/ml protaminesulfate (Sigma) for 24 hrs. Transduction efficiencies were calculatedwith pMX-GFP control virus. Cells were replated in fresh neuralexpansion medium. Two days after infection, the cells were furthersubcultured on irradiated MEFs in ESC medium containing LIF without anyfurther selection. Oct4-GFP-positive colonies were mechanicallyisolated, and individual cells were dissociated and subsequentlyreplated onto MEFs. The colonies were selected for expansion.

EXAMPLE 3: QRT-PCR ANALYSIS

Total RNA was extracted from cells using the MiniRNeasy Kit (QiagenGmbH, Hilden, Germany; http://www.qiagen.com) according to themanufacturer's instructions. Complementary DNA synthesis was performedwith the High Capacity cDNA Archive Kit (Applied Biosystems GmbH,Darmstadt, Germany; http://www.appliedbiosystems.com) following themanufacturer's instructions with a down-scaled reaction volume of 20 μl.Transcript levels were determined using the ABI PRISM Sequence DetectionSystem 7900 (Applied BioSystems) and the ready-to-use 5′-nucleaseAssays-on-Demand. For each real-time amplification, the template wasequivalent to 5 ng of total RNA. Measurements were done in triplicate; aRT⁻ blank of each sample and a no-template blank served as negativecontrols. Amplification curves and gene expression were normalized tothe housekeeping gene Hprt, used as internal standard.

Oligonucleotides were designed by the Taqman Assay-on-Demand for thedetection of the following genes: Pou5f1 (Oct3/4) (Mm00658129_gH), Sox2(Mm00488369_s1), c-Myc (Mm00487803_m1), Klf4 (Mm00516104_m1) B-Act(Mm00607939_s1), and Hprt1 (Mm00446968_m1). Oligos for the detection ofNanog and the viral sequences were custom-designed. Quantification wasnormalized on the endogenous Hprt gene within the log-linear phase ofthe amplification curve obtained for each probe/primers set using theΔΔCt method (ABI PRISM 7700 Sequence Detection System, user bulletin#2).

Primer sequences for viral-specific qRT-PCR pMXs-Oct4 PF: (SEQ ID NO:17) 5′-TGGTACGGGAAATCACAAGTTTG, PR: (SEQ ID NO: 18)5′-GTCATAGTTCCTGTTGGTGAAGTTCA, Probe: (SEQ ID NO: 19)5′-6FAM-CTTCACCATGCCCCTCA-MGB pMXs-Sox2 PF: (SEQ ID NO: 20)5′-GTGTGGTGGTACGGGAAATCAC, PR: (SEQ ID NO: 21) 5′-TTCAGCTCCGTCTCCATCATG,Probe: (SEQ ID NO: 22) 5′-6FAM-TGTACAAAAAAGCAGGCTTGT-MGB pMXs-Klf4 PF:(SEQ ID NO: 23) 5′-GTGTGGTGGTACGGGAAATCA, PR: (SEQ ID NO: 24)5′-CGCGAACGTGGAGAAGGA, Probe: (SEQ ID NO: 25)5′-6FAM-CTTCACCATGGCTGTCAG-MGB pMXs-cMyc PF: (SEQ ID NO: 26)5′-TGGTACGGGAAATCACAAGTTTG, PR: (SEQ ID NO: 27)5′-GTCATAGTTCCTGTTGGTGAAGTTCA, Probe: (SEQ ID NO: 28)5′-6FAM-CTTCACCATGCCCCTCA-MGB Nanog PF: (SEQ ID NO: 29)5′-AACCAGTGGTTGAATACTAGCAATG, PR: (SEQ ID NO: 30) 5′-CTGCAATGGAT GCTGGGATACT, Probe: (SEQ ID NO: 31) 5′-6FAM-TTCAGAAGGGCTCAGCAC-MGB

EXAMPLE 4: MICROARRAY ANALYSIS

The microarray study was carried out using Affymetrix Mouse Genome 4302.0 GeneChip arrays (Affymetrix, Santa Clara, Calif.) essentially asdescribed before (Ruau, D. et al., (2008), Stem Cells). Briefly, totalRNA was extracted from cells with RNAeasy kit including DNAse digestion(Qiagen, Hilden, Germany). Biotin-labelled cRNA was obtained from 3 μgof total RNA with the GeneChip One-Cycle labelling kit (Affymetrix).Fifteen micrograms of cRNA were fragmented and hybridized to Affymetrix430 2.0 GeneChip arrays at 45° C. for 16 hrs. DNA chips were washed,stained and scanned using an Affymetrix Fluidics device and GCS3000scanner, and the images obtained were analyzed using the GCOS software.The experiment was performed in triplicates for the ESCs and iPS cellsand in duplicates for the NSCs. Normalization was calculated with RMAalgorithm (Irizarry, R. A. et al., (2003), Nucleic Acids Res 31, e15)implemented in BioConductor.

EXAMPLE 5: IN VITRO DIFFERENTIATION OF IPS CELLS

Oct4-GFP cells were harvested by FACS analysis and used for in-vitrodifferentiation in embryoid bodies (EBs), which was performed withhanging drop in ESC medium without LIF. After 3 days, EBs were platedonto gelatine-coated 4-well dishes for another 10 days. The cells werestained with anti-Tuj1 antibody (1:100; Chemicon), anti-α-fetoprotein(AFP) antibody (1:100; R&D Systems), or anti-Flk1 antibody (1:100; R&DSystems).

EXAMPLE 6: WESTERN BLOT ANALYSIS, SSEA-1 AND AP STAINING

Total cell lysates (2×10⁶) prepared from the ESC and NSC were subjectedto western blot analysis for expression of Oct4 (Santa Cruz), Sox2(Santa Cruz), Klf4 (Abcam), and c-Myc (Abcam). β-actin expression levelsin all the samples were used as loading control (Abcam).

SSEA-1 and alkaline phosphatase (AP) staining was performed with the ESCell Characterization Kit (Chemicon) according to the manufacturer'sprotocol.

EXAMPLE 7: TERATOMA FORMATION

iPS cells and NSCs cells (1.5×10⁶ cells/mice) were injectedsubcutaneously into the dorsal flank of nude mice. Four weeks after theinjection, teratomas that had formed were fixed overnight in 4% PFA andembedded in paraffin. Sections were stained with haematoxylin and eosindyes.

EXAMPLE 8: CHIMERA FORMATION

iPS cells were aggregated and cultured with denuded post-compacted8-cell-stage mouse embryos. Briefly, 2-cell-stage embryos were flushedfrom mice [(C57BL/6×C3H) F1 females×CD1 males] at 1.5 dpc and placed inM2 medium and cultured overnight in KSOM medium with 0.1% BSA overnightto 8-cell stage. Clumps of loosely connected iPS cells (10-20 cells)from short trypsin-treated day-2 cultures were selected and transferredinto microdrops of KSOM medium with 10% FCS under mineral oil; eachclump was placed in a depression in the microdrop. Meanwhile, batches of30 to 40 embryos were briefly incubated with acidified Tyrode's solutionuntil the zona pellucida had disintegrated. A single embryo was placeonto the clump. All aggregates were assembled in this manner, andcultured at 37° C. in an atmosphere of 5% CO₂ in air. After 24 hours ofculture, the majority of the aggregates had formed blastocysts. A totalof 64 aggregated blastocysts (2.5 dpc) were transferred into the uterinehorns of five pseudopregnant mice (CD-1 background).

EXAMPLE 9: REPROGRAMMING OF HUMAN NEURAL STEM CELLS BY OCT4

hNSCs that derived from human fetal brain tissue were expanded inserum-free NSC medium as described previously (cf. FIG. 5A) (Kim et al.,Exp Neurol 199, 222 (2006); Park et al., Nat Biotechnol 20, 1111(2002)). hNSCs were first infected with pMXs encoding human OCT4 andKLF4 (2F) or OCT4 (1F). Then, infected hNSCs were maintained in NSCmedium (Kim et al., Exp Neurol 199, 222 (2006)) for up to 7 days. Day 8post-infection, the cells were replated onto feeder cell layers in hESCmedium containing 10 ng/ml bFGF and MEF-conditioned medium (CM) in a 1:1ratio which culture continued to grow until the hESC-like coloniesappeared. Within 10-11 weeks post-infection, the hESC-like iPS colonieswere identified but the centre of the colonies still appears like aneural rosette (cf. FIG. 5B). The colony grew larger exhibiting typicalhESC-like morphology within another 5-6 days but still the neuralrosettes remain in the center of the colony (cf. FIG. 5C). The neuralrosettes are removed from the colony. Then, a piece of the colony wastransferred on a feeder cell layer by mechanical isolation (cf. FIG.5D). We successfully established two clones out of three hESC-likecolonies by picking from OCT4 infected hNSCs (1F hNiPS clone A and C,reprogramming efficiency 0.02%). Otherwise, we also established 3 clonesout of five hESC-like colonies in 2F-infected hNSCs (2F hNiPS A, B andC, reprogramming efficiency, 0.15%) within 7-8 weeks post-infection. Allof which could be expanded in hESC culture condition. The 1F hNiPS cellswere morphologically similar to hESCs and stained positive for alkalinephosphatase (cf. FIGS. 5E and F). Immunofluorescence staining confirmedthat 2F and 1F hNiPS cells uniformly expressed the pluripotency markers,including OCT4, SSEA4, TRA-1-60 and TRA-1-81 (cf. FIG. 5G). Theseresults demonstrate that human iPS cells can be generated from hNSCs byOCT4 and KLF4 as well as OCT4 alone.

Next, we tested mRNA expression levels of pluripotency marker genes inthese iPS cells at molecular level by quantitative RT-PCR analysis. 2Fand 1F hNiPS cells endogenously expressed the hESCs-specific markers,were similar to H9 and H1 hESCs and were markedly up-regulated comparedwith parental hNSCs (cf. FIG. 6A). Genotyping PCR showed 1F hNiPS cloneshave an OCT4 transgene only and 2F hNiPS clones have OCT4 and KLF4transgenes in the genome. We also confirmed that the expression level oftransgenic OCT4 or KLF4 was significantly silenced in 2F and 1F hNiPSclones, except the OCT4 expression from 2F hNiPS clone B. Southern blotanalysis confirmed the integration of the OCT4 transgene in 2F and 1FhNiPS clones. To exclude the possibility that iPS clones arose throughcontamination from hESCs in the laboratory, DNA fingerprinting analysiswas performed and confirmed that hNiPS cells precisely correlate to thedonor hNSCs (cf. Table 2).

To confirm epigenetic remodelling of the OCT4 and NANOG promoters fromreprogrammed cells, we performed bisulfite sequencing to determine thedemethylation of both promoters. OCT4 and NANOG promoter regions weredemethylatd in 2F and 1F hNiPS cells relative to the donor hNSCs andwere similar to hESCs. Taken together, hNSCs can be reprogrammed intoiPS cells that similar to hESCs at molecular level by transduction ofOCT4 alone.

Next, we tested in vitro pluripotency of 2F and 1F hNiPS cells byembryoid body (EB) differentiation and direct differentiation. hNiPScells readily differentiated into endoderm (AFP), medoderm (a-SMA) andectoderm (Tuj1) by EB differentiation (cf. FIG. 5A) and we confirmed theexpression of all three germ layer makers from direct differentiation byquantitative RT-PCR analysis (cf. FIG. 7B). To confirm in vivopluripotency of these human iPS cells, the cells were subcutaneouslytransplanted into severe combined immunodeficient (SCID) mice. After 6-8weeks injection, 2F and 1F hNiPS cells gave rise to teratomas containingall three germ layers, including respiratory tract, skeletal muscle,cartilage and neural epithelium (cf. FIG. 8A). These results indicatethat 2F and 1F hNiPS cells have a pluripotency in vitro and in vivoalike hESCs.

Finally, we performed global gene expression analysis on hNSC, 2F and 1FhNiPS cells derived from hNSCs, H9 and H1 hESCs by cDNA microarrays.Heat map showed that 2F and 1F hNiPS cells similar to hESCs, otherwiseparental hNSCs are isolated from pluripotent populations (cf. FIG. 8B,left panel) and hierarchical clustering analysis showed that hNiPS cellsclustered with hESCs and were distinct from parental hNSCs (cf. FIG. 8B,right panel). Scatter plots analysis showed that hNiPS cells aresignificantly more similar to hESCs as like between different hESCs thanparental hNSCs (cf. FIG. 8C). 1F and 2F hNiPS cells also show similaritywith H1 hESCs. These data indicate that hNiPS cells are similar to hESCsin global gene expression profiles. Our results demonstrated 1F and 2FhNiPS cells closely resemble hESCs in molecular level and pluripotency.

TABLE 2 STR analysis of hNSCs and hNiPS cells 2F NhiPS 1F NhiPS Genomicloci H9 hESCs hNSCs A B C A C Amelogenin X; X X; Y X; Y X; Y X; Y X; YX; Y CSF1PO 11; 11 11; 13 11; 13 11; 13 11; 13 11; 13 11; 13 D13S317 9;9  8; 11  8; 11  8; 11  8; 11  8; 11  8; 11 D16S539 12; 13 9; 9 9; 9 9;9 9; 9 9; 9 9; 9 D18S51 13; 13 15; 16 15; 16 15; 16 15; 16 15; 16 15; 16D21S11 30; 30 31; 32 31; 32 31; 32 31; 32 31; 32 31; 32 D3S1358 13; 1616; 16 16; 16 16; 16 16; 16 16; 16 16; 16 D5S818 11; 12  7; 12  7; 12 7; 12  7; 12  7; 12  7; 12 D7S820  9; 11 11; 11 11; 11 11; 11 11; 1111; 11 11; 11 D8S1179  8; 14 12; 14 12; 14 12; 14 12; 14 12; 14 12; 14FGA 26; 28 23; 24 23; 24 23; 24 23; 24 23; 24 23; 24 Penta D  9; 13 11;12 11; 12 11; 12 11; 12 11; 12 11; 12 Penta E 11; 14 11; 18 11; 18 11;18 11; 18 11; 18 11; 18 TH01 9; 9 7; 7 7; 7 7; 7 7; 7 7; 7 7; 7 TPOX 10;11 8; 8 8; 8 8; 8 8; 8 8; 8 8; 8 vWA 17; 17 17; 17 17; 17 17; 17 17; 1717; 17 17; 17

Material and Methods: Cell Culture

Human NSCs were derived from the telencephalon (HFT13), established aspreviously described (Kim et al., Exp Neurol 199, 222 (2006)). Briefly,Telencephalon tissue was freshly dissected, dissociated in 0.1% trypsinfor 30 min and seeded into 10 cm plates at a density of 200,000 cells/mlin NSC medium. These cells were cultured in NSC medium as previouslydescribed (Kim et al., Exp Neurol 199, 222 (2006); Park et al., NatBiotechnol 20, 1111 (2002)). Human ES and iPS cells were maintained onmitomycin C-treated CF1 mouse feeder layers (Millipore) in human ESCmedium, which contains knockout DMEM (Invitrogen) supplemented with 20%knockout serum replacement (Invitrogen), 1 mM L-glutamine, 1%non-essential amino acids, 0.1 mM β-mercaptoethanol,penicillin/streptomycin and 10 ng/ml human basic fibroblast growthfactor (bFGF) (Invitrogen) as previously described (Takahashi et al.,Cell 131, 861 (2007)).

Induction of 1F hNiPS and 2F hNiPs Cells

The pMX-based retroviral vectors encoding the human cDNAs of OCT4 andKLF4 (Takahashi et Yamanaka, Cell 126, 663 (2006)) were cotransfectedwith packaging-defective helper plasmids into 293 cells using Fugenetransfection reagent (Roche) to produce vesicular stomatitis virus (VSV)G protein pseudotyped virus as previously described (Zaehres et Daley,Methods Enzymol 420, 49 (2006)). Viral supernatants were collected andconcentrated by ultracentrifugation 48 h post-transfection to infecthuman NSCs. For generation of iPS cells, human NSCs were seeded at adensity of 5×10⁴ cells per 6-well plate and incubated withvirus-containing supernatants for OCT4 or OCT4 and KLF4 supplementedwith 6 μg/ml protamine sulfate (Sigma) for 24 h. On the next day, themedium was replaced with fresh NSC medium at 1 d post-infection andmaintained up to 7 d post-infection. Cells were further cultured inhuman ESC medium from 8 d post-infection. The iPS colonies weremechanically isolated at 2 month or 2.5 month post-infection and weresubsequently replated and maintained on CF1 mouse feeder layers(Millipore) in human ESC medium.

Quantitative RT-PCR

Total RNA was isolated from bulk cell culture samples or hand-pickedundifferentiated colonies using RNeasy columns (Qiagen) with on-columnDNA digestion. cDNA was produced using oligo-dT₁₅ priming and M-MLVreverse transcriptase (USB) according to the manufacturer's instructionsat 42° C. for 1 h. About 50 ng of total RNA equivalent was typicallyused as template in 20 μl SYBR Green PCR reactions (40 cycles of 15″ 95°C./60″ 60° C. on Applied Biosystems 7300 instrumentation) thatadditionally contained 0.375 μM of each primer and 10 μl of SYBR GreenPCR mix (ABI). All primers used were confirmed to amplify the predictedproduct at close-to-optimal efficiency without side products. Primersequences are given in Table 3. Relative expression levels werecalculated using the comparative Ct method, based on biological controlsamples and two housekeeping genes for normalization. Error bars reflectstandard errors arising from biological replicates (marker geneexpression data) or from using independent housekeeping genes fornormalization (transgene silencing data).

Global Gene Expression Analysis

For transcriptional analysis, 400 ng of total DNA-free RNA was used asinput for labelled cRNA synthesis (Illumina TotalPrep RNA AmplificationKit—Ambion) following the manufacturer's instructions (IVT: 10 h).Quality-checked cRNA samples were hybridized as biological or technicalduplicates for 18 h onto HumanRef-8 v3 expression BeadChips (Illumina),washed, stained, and scanned following guidelines and usingmaterials/instrumentation supplied/suggested by the manufacturer. Themicroarray data are available from the GEO (Gene Expression Omnibus)website under accession number GSE GSE15355.

Bisulfite Sequencing

Genomic DNA was isolated from bulk cell culture samples or hand-pickedundifferentiated colonies using DNeasy columns (Qiagen). 300 ng was usedas input for bisulfite conversion (EpiTect Bisulfite Kit—Qiagen). 50 ngof converted DNA was used as template for conventional nested PCRsamplifying 467 and 336 bp regions of the OCT4 and NANOG promoters,respectively. Primers were specific for conversion of the sense DNAstrand and are given in Table 3. Purified PCRs were TA-cloned intopCR2.1-TOPO (Invitrogen). Insert sequences of randomly picked cloneswere analyzed using the BiQ Analyzer program, following its qualitycheck-based suggestions to drop individual clones if appropriate. Datafrom one CpG site at position +20 relative to the OCT4 translation startcodon is not shown as it was uninformative.

Short Tandem Repeat (STR) Analysis

Genomic DNA was isolated from cultured cell samples using DNeasy columns(Qiagen). This was used as template for STR analysis employing thePowerPlex 16 System (Promega) and ABI PRISM instrumentation. Numbersshown denote by lengths of the 15 autosomal fragments. The analysis wascarried out at Eurofins Medigenomix, Martinsried, Germany.

Teratoma Formation

hNiPS cells and hNSCs (3-5×10⁶ cells/mice) were injected subcutaneouslyinto the dorsal flank of SCID mice. Teratomas were fixed in 4% PFAovernight and embedded in paraffin after 6-8 weeks injection. Sectionswere stained with haematoxylin and eosin dyes.

Alkaline Phosphatase (AP) and Immunofluorescence Staining

Alkaline phosphatase (AP) staining was performed with the ES CellCharacterization Kit (Chemicon) according to the manufacturer'sprotocol. Immunofluorescence staining was performed using the followingprimary antibodies: AFP (Sigma, 1:100), a-SMA (Sigma, 1:50), TuJ1(Chemicon, 1:500), OCT4 (Santa Cruz, 1:200), SSEA4 (Chemicon, 1:200),TRA-1-60 (Chemicon, 1:200), TRA-1-81 (Chemicon, 1:200).

TABLE 3 Primers for Real-time PCR and Bisulfite sequencing. Real-timePCR Primers Gene Forward primer (5-3′) Reverse primer (5′-3′) ACTBTCAAGATCATTGCTCCTCCTGAG ACATCTGCTGGAAGGTGGACA AFP AGCAGCTTGGTGGTGGATGACCTGAGCTTGGCACAGATCCT CDH1 (E-CAD) TTGAGGCCAAGCAGCAGTACAATCCAGCACATCCACGGTGA CDX2 TCACTACAGTCGCTACATCACCATCTTAACCTGCCTCTCAGAGAGCC DNMT3B GCTCACAGGGCCCGATACTTGCAGTCCTGCAGCTCGAGTTTA DPPA4 TGGTGTCAGGTGGTGTGTGG CCAGGCTTGACCAGCATGAAFGF2 GGCAAGATGCAGGAGAGAGGA GCCACGTGAGAGCAGAGCAT FOXF1AAAGGAGCCACGAAGCAAGC AGGCTGAAGCGAAGGAAGAGG GAPDHCTGGTAAAGTGGATATTGTTGCCAT TGGAATCATATTGGAACATGTAAACC GATA6TGTGCGTTCATGGAGAAGATCA TTTGATAAGAGACCTCATGAACCGACT GDF3TTGGCACAAGTGGATCATTGC TTGGCACAAGTGGATCATTGC HAND1 TCCCTTTTCCGCTTGCTCTCCATCGCCTACCTGATGGACG KLF4 endo ACAGTCTGTTATGCACTGTGGTTTCACATTTGTTCTGCTTAAGGCATACTTGG KLF4 viral GTCGGACCACCTCGCCTTACTTTATCGTCGACCACTGTGCTG LIN28 GGAGGCCAAGAAAGGGAATATGAAACAATCTTGTGGCCACTTTGACA MYC CCAGCAGCGACTCTGAGGA GAGCCTGCCTCTTTTCCACAGNANOG CCTGTGATTTGTGGGCCTG GACAGTCTCCGTGTGAGGCAT NCAM1TCATGTGCATTGCGGTCAAC ACGATGGGCTCCTTGGACTC OCT4 endoGGAAGGAATTGGGAACACAAAGG AACTTCACCTTCCCTCCAACCA OCT4 viralGGCTCTCCCATGCATTCAAAC TTTATCGTCGACCACTGTGCTG SOX17 TTCGTGTGCAAGCCTGAGATGGTCGGACACCACCGAGGAA SOX2 TGGCGAACCATCTCTGTGGT CCAACGGTGTCAACCTGCAT TDGF1(Cripto) GGGATACAGCACAGTAAGGAGCTAA CACAAAAGGACCCCAGCATG ZNF206TCACCATGGCCAGAGGAGAG GCAGGCCACGCCTTATTCTC ZNF589 TCGGGTGGCTAAATTAGATCCAGCCCAAGGGAGTAAGGCAAACTG Primers for bisulfite sequencing Gene Forwardprimer (5′-3′) Reverse primer (5′-3′) OCT4 outerGAGGATAGGAATTTAAGATTAGTTTGGGTA AAATCCCCCACACCTCAAAACCTAACCCAA OCT4 innerGAGGTTGGAGTAGAAGGATTGTTTTGGTTT CCCCCCTAACCCATCACCTCCACCACCTAA OCT4 innerunconverted GAGGCTGGAGCAGAAGGATTGCTTTGGCCCCCCCCCTGGCCCATCACCTCCACCACCTGG NANOG outer TTAGTTTTTAGAGTAGTTGGGATTATAGAATAATAACATAAAACAACCAACTCAATCCA NANOG inner TGGTTAGGTTGGTTTTAAATTTTTGAACCCACCGTTATAAATTCTCAATTA NANOG inner unconvertedTGGCCAGGCTGGTTTCAAACTCCTG GACCCACCCTTGTGAATTCTCAGTTA

Southern Blot Analysis

BamHI digested genomic DNA from 1F hNiPS, hNSC and 2F hNiPS cells wereseparated on a 0.8% agarose gel and transferred to Biodyne B nylonmembrane (PALL Life Sciences). DNA was hybridized with a 32P labeledfragment of OCT4 (Eco81I (Saul) human OCT4 cDNA fragment) using theDecaLabel™ DNA Labeling Kit (Fermentas). Labeled Lambda HindIII digestedDNA served as a marker.

In Vitro Differentiation of Human iPS Cells

For immunocytochemistry, embryoid bodies (EBs) were generated from iPScells with the hanging drop method in MEF-conditioned medium. After 5days, EBs were transferred to gelatin-coated plate and subsequentculturing for another 14 days in knockout DMEM (Invitrogen) supplementedwith 20% FBS, 1 mM L-glutamine, 1% non-essential amino acids, 0.1 mMβ-mercaptoethanol, and penicillin/streptomycin. For qRT-PCR, iPScolonies were mechanically isolated and replated on Matrigel-coatedplate in MEF-conditioned medium. After 2 d, medium replaced with eachthree germ layer differentiation medium. For endoderm differentiation,the cells maintained in RPMI1640 medium supplemented with 2% FBS, 100ng/ml Activin A (R&D Systems), L-glutamine, and penicillin/streptomycinfor 3 weeks (Huangfu et al., Nat Biotechnol 26, 1269 (2008)). Formesoderm differentiation, knockout DMEM supplemented with 100 uMascorbic acid (Sigma), 20% FBS, 1 mM L-glutamine, 1% non-essential aminoacids, 0.1 mM β-mercaptoethanol, and penicillin/streptomycin for 3 weeks(Aasen et al., Nat Biotechnol 26, 1276 (2008)). For ectodermdifferentiation, the cells maintained in N2B27 medium for 7 days and themedium replaced with N2 medium supplemented with 10 ng/ml bFGF2(peprotech), 100 ng/ml Sonic Hedgehog (R&D Systems), 10 ng/ml PDFG (R&DSystems), L-glutamine, and penicillin/streptomycin for 2 weeks. Themedium was changed every other day. Primer sequences are given in Table3.

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1. A method of generating an induced pluripotent stem (iPS) cellcomprising the step of introducing into a target cell one or two codingsequences each giving rise upon transcription to a factor thatcontributes to the reprogramming of said target cell into an inducedpluripotent stem cell and selected from Oct3/4 or a factor belonging tothe Myc, Klf and Sox families of factors, wherein the target cellendogenously expresses at least one of the factors that are not encodedby the coding sequences to be introduced and selected from Oct3/4 orfactors belonging to the Myc, Klf and Sox families of factors, andwherein the cell resulting from the introduction of the one or twocoding sequences expresses the combination of factor Oct3/4 and at leastone factor of each family of factors selected from the group of Myc, Klfand Sox.
 2. The method of claim 1, wherein the factors belonging to thefactor families of Myc, Klf and Sox and endogenously expressed by orencoded by the coding sequences to be introduced into the target cellare selected from the group consisting of 1-Myc, n-Myc, c-Myc, Klf1,Klf2, Klf4, Klf15, Sox1, Sox2, Sox3, Sox15 and Sox18.
 3. The method ofclaim 1, wherein the target cell does not endogenously express said atleast one of the factors encoded by the one or two coding sequences tobe introduced into said target cell.
 4. The method of claim 1, whereinthe target cell is a multipotent stem cell.
 5. The method of claim 4,wherein the multipotent stem cell is an ectodermal cell.
 6. The methodof claim 1, wherein the target cell is a neural stem cell (NSC).
 7. Themethod of claim 6, wherein the coding sequence to be introduced encodesthe factor Oct3/4.
 8. The method of claim 6, wherein the two codingsequences to be introduced encode factors Oct3/4 and c-Myc or Oct3/4 andKlf4.
 9. The method of claim 7, wherein the target cell endogenouslyexpresses the factors c-Myc, Klf4 and Sox2.
 10. The method of claim 9,wherein the target cell endogenously expresses the factors c-Myc, Klf4and Sox2 at levels at least 10-fold lower or at most 10-fold higher ascompared to the corresponding expression levels in embryonic stem cellsof the same genus as the target cell.
 11. The method of claim 7, whereinthe target cell is a murine neural stem cell.
 12. An induced pluripotentstem cell generated by the method of claim
 1. 13. A method ofidentifying a compound that contributes to the reprogramming of a targetcell into an induced pluripotent stem cell comprising the steps of: (a)reprogramming a target cell according to the method of claim 1, whereinone coding sequence to be introduced is replaced by the compound to betested; and (b) assessing whether iPS cells are formed in the presenceand absence of the compound to be tested, wherein the formation of iPScells from target cells in which the compound to be tested has beenintroduced is indicative of the compound contributing to thereprogramming of a target cell into an induced pluripotent stem cell.14. A method of generating a transgenic non-human animal comprising thesteps of: (a) introducing the induced pluripotent stem cell generated bythe method of claim 1 into a non-human preimplantation embryo; (b)transferring the embryo of step (a) into the uterus of a femalenon-human animal; and (c) allowing the embryo to develop and to be born.15. A transgenic non-human animal generated by the method of claim 14.16. A composition comprising an iPS cell generated by the method ofclaim 1 for gene therapy, regenerative medicine, cell therapy or drugscreening.